10.11 The flow of nutrients: transport and translocation

The plasma membrane is a lipoidal layer separating the aqueous ‘bubble’ of
the cell from its aqueous surroundings. This separation is not complete or
absolute as the cell must exchange chemicals with the environment; removing
excretion products and absorbing nutrients. However, only molecules which
dissolve readily in lipid are able to penetrate the membrane without assistance.
Since the vast majority of molecules the cell needs to transfer across the
membrane are hydrophilic rather than lipophilic, plasma membranes have evolved a
range of associated transport systems that permit selective
communication between the two sides of the membrane. This selectivity permits
the cell to exercise considerable control over its interaction with the
environment.

In an infinite solution, molecules of solute can move within the solution in
two ways. Whole volumes of solution may be transported from place to place,
taking solute molecules with them. This is bulk flow or mass flow
and results from such things as convection flows and other large-scale
disturbances within the solution. As far as living organisms are concerned, bulk
flow may be achieved through cytoplasmic streaming, transpiration streams and
similar processes. Although it is becoming clear that multiple motor proteins
may work together to drive intracellular transport of organelles (Rai et al.,
2013; Pathak & Mallik, 2017), we must emphasise that what we are describing here
is different from, and additional to, the vesicle and vacuolar traffic described
in Chapter 5, above (Section 5.10 The
endomembrane systems; Section 5.11
Cytoskeletal systems; and Section
5.12Molecular motors). Interestingly, strongly-bound kinesins fail to work
collectively, whereas detachment-prone dyneins team up. It seems that leading
dyneins in a team take short steps, while trailing dyneins take larger steps;
the dyneins consequently bunch together, which shares the load more effectively
and bonds the motor proteins more tenaciously to the microtubule. Even though
such behaviour allows vesicle and vacuolar traffic to sustain the most rapid of
extension growth rates in filamentous fungi, the bulk flow we describe here is
more likely to be associated with distribution of materials (whether in solution
or not) over multicellular or intercellular dimensions
than with transfers via the endomembrane and cytoskeletal systems.

The second mode of solute movement is diffusion, where
random thermal motion at the molecular level causes all solute molecules to move
continuously. If the solution is completely homogeneous then any molecules which
move out of a particular unit volume will be replaced by an identical number
moving into that unit volume and the exchange of solute molecules will not be
detectable. On the other hand, if there is a concentration gradient within the
solution there will be a net flow of solute molecules from the high
concentration end of the gradient, towards the low concentration end. Note that
this gradient can be a chemical gradient of uncharged molecules (e.g. a sugar),
an electrical gradient of a charged ion (e.g. K+) or a combination of
the two. This diffusion process is extremely relevant to the behaviour of cells,
since there is likely to be a concentration gradient across the
plasma membrane for just about every solute of importance to the cell.

To traverse the biological membranes a solute must leave the aqueous phase
for the lipoidal environment of the membrane, traverse that, and then re-enter
the aqueous phase on the other side of the membrane. Unaided simple diffusion of
molecules across biological membranes depends considerably on their solubility
in lipids. There are exceptions to this generalisation, though, as some small
polar molecules (such as water) enter cells more readily than would be expected
from their solubility in lipid. They behave as though they are traversing the
membrane by simple diffusion through gaps or pores which are
transiently generated by random movements of the acyl chains of the membrane
phospholipids. Transfer of these materials (like that of molecules which are
soluble in the lipid bilayer of the membrane, such as O2 and CO2)
depend on simple diffusion. Their rate of movement is then
proportional to the concentration differential on the two sides of the membrane
and the direction of movement is from the high to the low concentration side. No
metabolic energy is expended and no specific membrane structures are involved in
this mode of transfer, but net transfer ceases when the transmembrane
concentrations equalise.

Only a minority of compounds pass through biological membranes in vivo
by simple diffusion; the vast majority of metabolites that the cell needs to
absorb or excrete are too polar to dissolve readily in lipid and too large in
molecular size to make use of transient pores. To cope with these circumstances
the membrane is equipped with solute transport systems. This
applies to intracellular membranes bounding compartments within the cell as well
as to the plasma membrane. The essential component of any transport system is a
transporter molecule, a protein which spans the membrane and assists
transfer of the metabolite across the lipid environment of the membrane.

With both passive and active transporters, substrate translocation depends on
a conformational change in the transporter such that the substrate binding site
is alternately presented to the two faces of the membrane. These transporters
are transmembrane glycoproteins of around 500 amino acids
arranged into three major domains: 12 α-helices spanning the membrane, a highly
charged cytoplasmic domain between helices 6 and 7, and a smaller external
domain, between helices 1 and 2, which bears the carbohydrate moiety. Sequence
homology between the N- and C-terminal halves of the protein suggests that the
12 α-helix structure has arisen by the duplication of a gene encoding a 6-helix
structure. Ion channels are different as their polypeptide subunits form a
β-barrel containing a pore. One loop of the polypeptide is folded into the
barrel and amino acids of this loop determine the size and ion selectivity of
the channel. This transporter alternates between open and closed conformations.

If the transfer is passive with no requirement for metabolic
energy then the transport process is described as facilitated diffusion.
Such a process still depends upon a concentration differential existing between
the two sides of the membrane, transfer occurring ‘down the gradient’ (towards
the compartment which has the lower concentration). However, transfer is much
faster than would be predicted from the solubility of the metabolite in lipid,
the high rate of transfer depending on the fact that the transporter and the
transporter/metabolite complex are highly mobile in the lipid environment of the
membrane. The major differences from simple diffusion are that facilitated
diffusion exhibits:

high substrate specificity;

saturation kinetics.

Showing saturation kinetics means that as the concentration of the metabolite
being transported is increased, the rate of transport increases asymptotically
towards a theoretical maximum value at which all the transporters are complexed
with the metabolite being transported (i.e. transporters are saturated).

Facilitated diffusion can transport a specific substrate very rapidly; but
can only equalise the concentrations of the transported metabolite on the two
sides of the membrane. Yet in many cases the cell needs to transfer a metabolite
against its concentration gradient. The prime example will be where the
cell is absorbing a nutrient available at only a low concentration; if growth of
the cell is not to be limited by the external concentration of the nutrient, the
cell must be able to accumulate the nutrient to concentrations greater than
those existing outside. In which case an adverse gradient of concentration will
have to be established and maintained. Neither simple diffusion nor facilitated
diffusion can do this; to achieve it the cell must expend energy to drive the
transport mechanism. Such a process is called active transport.

Active transport is a transporter-mediated process in which movement of the
transporter/substrate complex across the membrane is energy dependent. The
transporter exhibits the same properties as a facilitated transport transporter
(saturation kinetics, substrate specificity, sensitivity to metabolic
inhibitors). In addition to these properties, active transport processes
characteristically transfer substrate across the membrane against a chemical
and/or electrochemical gradient, and are subject to inhibition by conditions or
chemicals which inhibit metabolic energy generation.

The mechanism is often a co-transport
in which the movement of an ion down its electrochemical gradient is coupled to
transport of another molecule against its concentration gradient. When the ion
and the transported substrate move in the same direction the co-transporter is
called a symport, whereas transporters which transport the two
in opposite directions are termed antiporters. The
electrochemical gradients, most usually of protons or K+
in fungi, are created by ion pumps in which hydrolysis of ATP
phosphorylates a cytoplasmic domain of the ion channel. Consequential
conformational rearrangement of the protein then translocates the ion across the
membrane and reduces the affinity of the binding site to release the ion at the
opposite membrane face. Dephosphorylation restores the pump to its active
conformation (and may translocate another ion or molecule in the opposite
direction).

Complex interactions occur in transport of anions, cations and
non-electrolytes; interactions which may depend on metabolic, chemical,
biophysical and/or electrochemical relationships between a number of different
molecular species and with the rest of metabolism. There are indications of what
might be called transport strategy in operation in most cells. Single uptake
systems are rarely encountered; dual or multiple systems are the norm, the
different components being suited to different environmental conditions the
organism may encounter. Multiple uptake systems inevitably result in complex
uptake kinetics which might be indicative of physically separate transport
transporters, each showing Michaelis-Menten kinetics (like the glucose
transporters in Neurospora), or of single molecules exhibiting kinetics
modulated by their environment (like the glucose transporter in Coprinopsis;
Moore & Devadatham, 1979; Taj Aldeen & Moore, 1982).

Whatever the physical basis, the regulatory properties of the components of
such ‘families’ of transport processes appear to be interlinked to ensure that
nutrient uptake is maintained at a reliable level whatever the variation in
substrate availability in the environment. Probably the most important
generalisation that can be made about transport processes, though, is that for
almost all of them the active extrusion of protons from the fungal cell seems to
be essential. The proton gradient so established provides for uptake of sugars,
amino acids and other nutrients by proton co-transport down the gradient, and is
directly involved in cation transport like the K+/H+
exchange or antiport. So, each fungus possesses multiple uptake systems
for most nutrients but the same basic process (active H+ extrusion)
energising most if not all.

A crucial point, which has not yet been taken into account, is that the
transport systems so far described will inevitably alter the solute
concentrations of the cell and thereby influence the movement of that
all-pervading nutrient, water. Water is a significant (even if often overlooked)
component of innumerable biochemical processes. For example, every hydrolytic
enzyme reaction uses a molecule of water, every
condensation reaction produces a molecule of water and
respiration of 1 g of glucose produces 0.6 g of water. The water relationships
of the fungal cell are an important aspect of its overall economy. Water
availability is determined by its potential energy; referred to as the
water potential, symbolised by the Greek letter psi (Ψ). Zero water
potential is the potential energy of a reference volume of free, pure water. The
water in and around living fungal cells will have positive or negative potential
energy relative to that reference state, depending on the effect(s) of osmotic,
turgor, matrix and gravitational forces. Water will flow spontaneously along a
water potential gradient, from high to low potentials, though in the normal
state for most fungi this will mean from a negative to a more negative
potential. The lower the water potential the less available is the water for
physiological purposes and the greater is the amount of energy that must be
expended to make the water available.

On the face of it, two things need to be considered. One is some sort of
compensation for change in the solute relationships of the cell resulting from
uptake of some substrate; such a process would further reduce the potential of
the cell water and increase the tendency of external water to influx. The other
is to provide the cell with a means to regulate its water uptake even though the
external water potential is uncontrollable. In fact, of course, these are just
two facets of the same problem. In either case the fungus must cope with water
potential stress and the evidence indicates that solute transport systems
provide the mechanism which permits this. The internal maintenance of
turgor pressure by movement of water across the membrane is related to
transport of ions across the membrane and to the breakdown of macromolecules and
biosynthesis of solutes. Inorganic ions usually make the greatest percentage
contribution to the osmotic potential of the protoplasm. The main ions involved
are K+ and Na+, with Cl- being moved to balance
the cation content. Some organic solutes also make major contributions,
including glycerol, mannitol, inositol, sucrose, urea and proline.

The most immediate response to water potential stress is change in cell
volume by the rapid flow of water into or out of the cell. The consequent change
in turgor affects the cell membrane permeability and electrical properties so
that the cell can restore the volume by transporting ions or other solutes
across the membrane and/or by synthesising solutes or by obtaining them by
degrading macromolecules. Response to water potential stress can be extremely
rapid. Experimentally this is particularly evident in fungal protoplasts, the
size of which alters soon after change in the solute concentration of the
suspending medium. Such behaviour attests to the ready permeability of the cell
membrane to water.

Polar water molecules can move across cell membranes
despite their lipid (hydrophobic) environment forming a natural barrier to
their transport; such transport is called osmosis,
which is just a special type of simple diffusion. Diffusion of water through
lipid sounds like a very unlikely event, and it is, but what drives it is
what drives all diffusion events, which is the relative concentrations of
the diffusing molecule at the ‘source’ and at the ‘sink’. In the case of
water molecules penetrating a lipid membrane, the concentration of water in
the lipid phase (the ‘sink’) will be extremely low
(the solubility of water in lipid is about 1 molecule of water per million
molecules of lipid), but the concentration in the aqueous phase (the
‘source’) will be extremely high [we’ll leave you to calculate the
concentration of water; remember a molar solution contains the
gram-molecular mass of a solute in one litre of solution, and that the mass
of a litre of water, by definition, is 1,000 g and the molecular mass of H2O
is 18. If you think all that’s too easy, Google Avogadro’s
number and work out how many water molecules there are in a
200 ml glass of water].

Such extreme diffusion gradients, with the additional
facts that the water molecule is very small and the surface area to volume
ratio of the cell is large, offset the very low permeability of the membrane
and allow water to diffuse through the lipid bilayer. That’s not the whole
story, of course, because in some membranes the water flux is too high to be
accounted for by simple diffusion alone. In such cases, water migrates by
facilitated diffusion (see
Section 5.13) through pores or channels provided by proteins called
aquaporins that form membrane-spanning complexes. Water moves
through these channels passively in response to osmotic gradients (Nehls &
Dietz, 2014).

The managed flow of water, coordinated with control of the wall synthetic
apparatus, must be a prime factor in controlling the inflation of fungal cells
which is responsible for many of the changes in cell shape which characterise
fungal cell differentiation. Turgor also contributes to flow along the fungal
hypha. As this is a filamentous structure, flow of water and solutes within the
hypha (i.e. translocation) is of enormous importance. Although our current view
of apical growth requires that fungi can organise rapid translocation and
specific delivery of various vacuoles and microvesicles, more general water flow
along the hypha is driven by a turgor gradient and solutes are translocated by
this turgor-driven bulk flow. Translocation of nutrients of all sorts in this
manner is of crucial importance to morphogenesis because it must be the main way
in which developing multicellular structures, such as a fruit body developing on
a vegetative colony, are supplied with nutrients and water. Translocation is
ably discussed by Jennings (2008; see his Chapter 14) and the mechanism can best
be illustrated by quoting his description of the way in which Serpula
lacrymans (the major timber decay organism in buildings in northern Europe)
translocates carbohydrate:

“Mycelium attacks the cellulose in the wood, producing glucose, which is
taken into the hyphae by active transport. Inside the hypha, glucose is
converted to trehalose, which is the major carbohydrate translocated. The
accumulation of trehalose leads to the hypha having a water potential lower
than outside. There is a flux of water into the hyphae and the hydrostatic
pressure so generated drives the solution through the mycelium. The sink for
translocated material is the new protoplasm and wall material produced at the
extending mycelial front. The mechanism of translocation in S. lacrymans
is thus the same as that now accepted for translocation in the phloem of
higher plants, namely osmotically driven mass flow. (Jennings, 2008; p. 459).”

By measuring the increase in volume of developing cord networks of
Phanerochaete velutina, Heaton et al. (2010) established that
hyphal (and cord) growth induces mass flows across the whole mycelial network.
This compensates for the fact that osmotically driven water uptake is often
distal from the tips, and results in a rapid global response to local fluid
movements. Coupling of growth and mass flow enables development of efficient,
and highly adaptive mycelial transport networks. Velocity of fluid flow in each
cord becomes a local signal that conveys information about the role of each cord
within the mycelium; cords carrying fast-moving or large fluid flows were
significantly more likely to increase in size than cords with slow-moving or
small currents.

Jennings' (2008) description
quoted above could be paraphrased to apply to
other circumstances by, for example, featuring nutrients other than
carbohydrates and/or alternative sinks, such as fruit bodies or particular
tissues in fruit bodies. Importantly, this bulk flow does not have to be
unidirectional within a tissue. Because the tissue is comprised of
a community of hyphae, different hyphae in that community may be
translocating in different directions simultaneously. Nutrients labelled
with different radioisotopes have been used to demonstrate that carbon is
translocated simultaneously in both directions along rhizomorphs of
Armillaria mellea. In mycorrhizas, carbon sources from the host plant
and phosphorus absorbed by the hyphae from the soil must move simultaneously
in opposite directions. Indeed, the flow of carbon in mycorrhizas must be
fairly complex as carbon can be transferred between two different plants
which are connected to the same mycorrhizal system. Although much remains to
be learned, there is clear evidence that nutrients (including water in that
category)
can be delivered over long distances through mycorrhizal
hyphal systems, and that the flows can be managed and targeted to specific
destinations in this ‘mycorrhizal transportome’ as circumstances demand
(Courty et al., 2016).